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WO1991009965A1 - Toxine amelioree d'elaboration d'immunotoxines - Google Patents

Toxine amelioree d'elaboration d'immunotoxines Download PDF

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Publication number
WO1991009965A1
WO1991009965A1 PCT/US1990/007464 US9007464W WO9109965A1 WO 1991009965 A1 WO1991009965 A1 WO 1991009965A1 US 9007464 W US9007464 W US 9007464W WO 9109965 A1 WO9109965 A1 WO 9109965A1
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Prior art keywords
lyspe40
cells
tfr
molecule
tumor
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PCT/US1990/007464
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English (en)
Inventor
Ira H. Pastan
David Fitzgerald
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Application filed by The United States Of America, Represented By The Secretary, United States Department Of Commerce filed Critical The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority to JP91502841A priority Critical patent/JPH04506976A/ja
Publication of WO1991009965A1 publication Critical patent/WO1991009965A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the parent application (06/911,227) teaches the production of reco binant proteins from modified Pseudomo- nas exotoxin (PE) gene fused with DNA sequences encoding a recognition protein for which a specific receptor exists on the cells. This was exemplified in the parent applica ⁇ tion with the construction and expression of an IL-2-PE fusion gene.
  • the present application relates to the production of improved toxins which are useful in con ⁇ structing more effective immunotoxins.
  • the invention provides an altered PE molecule, designated herein as Lys-PE40, that can be easily conjugated to an antibody with effective cytotoxicity toward target cells.
  • Figure 1 schematically illustrates the structure of plasmid pVC85L showing the presence of a T7 promoter, and OmpA signal sequence and domains II (translocation domain) and III (ADP ribosylation domain) of Pseudomonas exotoxi .
  • Figure 2A shows the results of SDS-PAGE analysis of LysPE40 pools at various stages of purification. 12.5% gels were stained with Coomassie blue. Lane 1, QMA concentrated culture medium proteins; Lane 2, Q Sepharose pool; Lane 3, Flow through material from Mono S; Lane 4, Mono S pool; Lane 5, TSK 250 pool. The positions of molecular weight standards are indicated.
  • B SDS-PAGE analysis of anti-TFR-LysPE40. Samples were applied on a 10% reducing polyacrylamide gel. Lane 1, ⁇ TFR-LysPE40; Lane 2, LysPE40; Lane 3, anti-TFR. Gels were stained with Coomassie blue.
  • Figure 3A demonstrates the inhibition of protein synthesis in A431 cells by anti-TFR-LysPE40.
  • Immunotoxin was added to the cells in the absence (*) and presence (o) of excess anti-TFR (tp ⁇ g/ml) for 18-24 hrs at 37°C.
  • B Cytotoxic activity of anti-Tac-LysPE40 on HUT102 cells.
  • Anti-Tac-LysPE40 was added at various concentrations to the indicated cell lines.
  • 70 ⁇ g/ml anti-Tac was added before the addition of the immunotoxin. 3 H leucine incorporation was measured as described in the text.
  • Figure 4 shows the blood levels of anti-TFR- LysPE40.
  • BALB/C mice or nude mice with A431 tumors were injected I.P. with 100 ⁇ g or 50 ⁇ g of anti-TFR-LysPE40.
  • Immunotoxin levels were measured in serum at different time periods. Results are average of two different experiments.
  • Figure 5 shows the effect of anti-TFR-LysPE40 on the growth of A 31 tumors in nude mice.
  • Mice were inject ⁇ ed with 3 x 10 6 A431 cells and treated with the immunotoxin as indicated. Mice were given four doses of 50 ⁇ g each on the indicated days: (#) days 2, 4, 6, 8; (A) days 9, 11, 13 and 15; ( ⁇ ) no treatment.
  • B Mice given four doses on days 5, 7, 9 and 11; ( ⁇ ) no treatment; (D) 5 ⁇ g; (#); 20 ⁇ g; (A), 50 ⁇ g; (A), single dose of 150 ⁇ g on day 5.
  • Figure 6 shows the gross appearance of subcutane ⁇ ous A431 tumors in nude mice with and without treatment with HB21-PE40.
  • mice were injected subcutaneously with 3 x 10 6 A431 cells on day 0. Without further treatment, the tumors were apparent as small bumps under the skin on day 5 (A; arrows). By day 15, tumors were large, often erupting through the skin surface (B) . Mice treated with HB21-PE40 on days 2, 4, 6 and 8 usually showed no develop ⁇ ment of tumor, demonstrated here as the absence of gross tumor on day 26 (C) . When mice with large tumors were treated with HB21-PE40 (on days 9, 11, 13 and 15), the tumors often began to shrink, collapsing inward on their necrotic centers (D; arrows).
  • Figure 7 shows the histological appearance of typical treated and control A431 subcutaneous tumors.
  • A431 tumors were removed at necropsy, fixed on formaldehyde and processed for routine paraffin embedding and sectioning, and staining with hematoxylin and eosin.
  • a section from a tumor removed from a mouse at day 15 is shown in A-4.
  • a similar section of a tumor removed from a mouse on day 19 that had been treated on days 9, 11, 13 and 15 with anti-TFR-LysPE40 is shown in B-B.
  • the cross- section shown in A shows that the majority of the untreat ⁇ ed tumor contains viable tumor cells (VT) with only small areas of necrosis (arrow).
  • VT viable tumor cells
  • A', A" and (B'B") are higher magnification fields from the margins of tumors demonstrating that the untreated tumor is mostly composed of homogeneous viable tumors (VT), whereas the treated tumor shows a rim of viable cells of variable thickness lying adjacent to a connective tissue capsule (ct).
  • the majority of the treated tumor shows large areas of central necrosis, and in some areas of the tumor margin all tumor cells are necrotic, whereas in other areas a viable tumor rim only a few cells thick remains (B", double arrow).
  • an improved Pseudomonas exotoxin of the type including a deletion in the receptor binding domain la of the native toxin, wherein the improvement comprises a recombinant PE mole ⁇ cule possessing at least one lysine residue in domain la of the PE molecule, which otherwise will be devoid of a lysine residue when having a deletion in the receptor binding domain la, said lysine residue providing an essential linkage for effective coupling of the recombi ⁇ nant PE to other molecules, such as antibodies and the like.
  • lysine residues are required for coupling PE to other molecules.
  • LysPE40 Since all 12 lysine residues of domain I are lost when domain I is deleted from PE, one of the three lysine residues in the other part of the molecule (for instance, domain III), must be used to couple PE40 to an antibody or another targeting molecule. However, when one of these three lysine resi ⁇ dues are used, a conjugate with low activity is obtained (Kondo et al, J. Biol. Chem. 263:9470, 1988). In order to overcome this problem and to obtain a conjugate with high activity, a new PE molecule was created by deleting most of domain I (residues 6-252) but maintaining one of the 12 lysine residues originally present in domain I. This new molecule, in accordance with the present invention, is designated herein as LysPE40.
  • the term "antibody” includes various forms of modified or altered antibodies, such as an intact immuno- globulin, an Fv fragment containing only the light and heavy chain variable regions, an Fab or (Fab) ' 2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody (Bird et al., 1988, Science 242, 424-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879-5883), and the like.
  • the antibody may be of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al. , 1984, Proc. Nat. Acad. Sci.
  • fusion protein of the present invention does not affect the normal functions of the untargeted cells to any appreciable degree or to any abnormal level.
  • the recombinant antibody-LysPE40 fusion protein may contain one polypeptide chain or two chains.
  • the example disclosed herein relates to the one chain case. To produce two chains, no a ino acid linker would be inserted between the V H and V L sequences. Instead a termination codon would be inserted after the V H sequence, and an initiation codon and ribosome binding sequence would be inserted before the V L sequence.
  • the V L and V H sequences will be followed respectively by part or all of the light and heavy chain constant regions, e.g., the whole kappa light chain constant region and the C H 1 domain of the heavy chain constant region, with or without the heavy chain hinge domain.
  • V L , V H and PE40 genes may occur in any order on the plasmid, hence the PE40 gene may be attached to either the 5' or 3' end of either the light or heavy chain gene.
  • the PE40 gene may be attached to either the 5' or 3' end of either the light or heavy chain gene.
  • additional modifications, deletions, insertions and the like may be made to the antibody and LysPE40 genes.
  • deletions or changes may be made in LysPE40 or in the linker connecting the antibody gene to LysPE40, in order to increase cytotoxicity of the fusion protein toward target cells or to decrease cytotoxicity toward cells without antigen for the antibody.
  • Plasmid pVC4 (derived from pJH4 by treating pJH4 with Sphl and Tthllll and relegating the large fragment containing the PE gene) was joined to an OmpA signal sequence as described by Chaudhary et al., PNAS 85, 2929- 2943, 1988, to produce pVC45.
  • Site directed mutagenesis is a convenient way to make deletions and it was used to create plasmid pJY85L which produces LysPE40 as shown in Figure 1.
  • a Hindlll/Sall fragment of the PE gene in pVC45 was cloned into M13, mpl9 and uracil containing single stranded DNA prepared by the method of Kunkel (kunkel, T.A. , PNAS 82, 488-492, 1985).
  • An oligonucleotide 36 nucleotides in length with the structure 5'CCAGGCTGCCGC- CCTTGAAAGCTTGGCGTAATCATG3' was synthesized which generated a large deletion in PE of amino acids 6 through 252 and retained a lysine residue between amino acids 5 and 253 to give a molecule with the sequence:
  • Hindlll/Sall fragment was excised from the replicative form of the mutant DNA in Ml3 and ligated with a 3.3 kb Hindlll/Sall fragment of pVC45 to give pJY85L which encodes a protein with the following struc ⁇ ture: Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly- Phe
  • This protein is processed at the ( 1 ) and the processed product secreted into the medium.
  • LysPE40 was found in and purified from the medium with a yield of greater than 1 mg per liter.
  • the sequence of amino acids at its amino terminus was found to be that predicted from the DNA sequence:
  • a deposit of plasmid pJY85L has been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., on December 18, 1989 under the accession number 68189.
  • the deposit shall be viably maintained, replacing it if it becomes non- viable, for the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public upon issuance of a patent from this application, without re ⁇ striction, in accordance with the provisions of the law.
  • the Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
  • BL21 ( DE3) cells were transformed with plasmid pVC85L and grown in LB medium containing 100 ⁇ g/ml ampi- cillin at 37°C.
  • LB medium containing 100 ⁇ g/ml ampi- cillin at 37°C.
  • absorbance 0.6 (650 nm) isopropyl-1- thio- ⁇ -D-galactopyranoside was added to a final concentra ⁇ tion of 1 mM. Cells were harvested 90 min later.
  • the culture medium was used as the source of LysPE40 because most of the protein was secreted into the medium. Purification of LvsPE40 Clarified culture medium, 25 liters, containing
  • LysPE40 was diluted four-fold with chilled deionized water and applied on a 10 x 5.5 cm silica-based anion exchange column (QMA, Waters) at a flow rate of 100 ml/min.
  • the column was washed with 0.05 M sodium phosphate buffer, pH7.0, and proteins were eluted with two liters of 0.25 M NaCl in the equilibration buffer.
  • LysPE40 from the QMA column was concentrated further by using Amicon YM30 membranes to 150 ml, dialyzed for 12-16 hours against 0.02 M Tris-HCl, pH7.6, centrifuged at 10,000 x g for 20 minutes and applied on a 2.5 x 14 cm Q-Sepharose column equilibrated with 0.02 M Tris-HCl, pH 7.6. Proteins were eluted with a 500 ml linear gradient of 0-0.5 M NaCl; LysPE40-containing fractions were detected by SDS PAGE, pooled, diluted 10-fold with 0.02 M Mes, pH 5.5, and loaded onto an FPLC Mono S 16/10 column equilibrated with 0.02 M Mes, pH 5.5.
  • LysPE40 bound to the column and was eluted by a 100 ml linear gradient of 0-0.5 M NaCl. It was further purified on TSK250 (22.5 x 600 mM) column (BioRad) using 0.2 M sodium phosphate buffer pH 7.0 containing 1 mM EDTA.
  • LysPE40 (2.5 mg/ml) in 0.2 M sodium phosphate buffer pH 7.0 containing 1 mM EDTA was mixed with a 3-fold molar excess of SMCC and incubated at room temperature (about 22°-24°C) for 30 minutes. Protein was separated from the unreacted cross linker on a PD10 column.
  • MoAb (HB21 or ⁇ Tac) (5-10 mg/ml) was mixed with a 3-fold molar excess of 2-iminothiolane-HCl in 0.2 M sodium phosphate buffer pH 8.0 containing 1 mM EDTA and incubated at 37°C for 1 hr. The derivatized antibody was separated from the reactants on a PD10 column.
  • TFR- LysPE40 which binds to human transferrin receptor (TFR)
  • Tac-LysPE40 which binds to 55 kD a subunit of human IL2 receptor.
  • BALB/C mice or nude mice bearing A431 subcutaneous tumors were injected with 100 ⁇ g or 50 ⁇ g of immunotoxin I.P. Blood was drawn at different time points and the level of the IT was assayed by incubating serum with A431 cells and measuring its effect on protein synthesis. A standard curve was made using anti-TFR-LysPE40. Antitumor activity of anti-TFR-LysPE40 in nude mice bearing a human epidermoid carcinoma
  • LysPE40 As mentioned before, the amino end of PE40 was altered so that it contained a lysine residue and an OmpA signal sequence. (The OmpA signal sequence was added primarily to direct the export of LysPE40 to the growth medium. )
  • the structure of LysPE40 and the plasmid encod ⁇ ing LysPE40 which is under the control of a T7 promoter is shown in Figure 1. Similar to PE40, LysPE40 was also secreted in the culture medium in large amounts. The purity of the protein at each purification step is shown in Figure 2. Material from the TSK 250 column, which was used as the final step, was found to be homogeneous when analyzed by SDS PAGE (Fig. 2, lane 5) as well as by immunoblotting with an antibody to PE (data not shown) . Typically, 2 mg of pure LysPE40 was obtained from one liter of culture.
  • the N-terminal sequence of the purified protein (LysPE40) was found to be Ala-Asn-Leu-Ala-Glu-Ala-Phe-Lys- Gly-Gly-Ser-Leu.
  • the purified protein had the exact sequence expected from the DNA sequence and processing occurred within the OmpA sequence ( Figure 1).
  • the N-terminal sequence of PE40 is Ala-Asn-Leu-Ala- Glu-Glu-Gly-Gly.
  • LysPE40 was chemically coupled to two different monoclonal antibodies, HB21 which binds to the human transferrin receptor (anti-TFR) (Haynes et al, J. Immunol 127:347, 1981) and anti-Tac which binds to the 55 kDa subunit of human interleukin 2 receptor (Uchiyama et al, . Immuno. , 126:1393, 1981).
  • anti-TFR human transferrin receptor
  • anti-Tac which binds to the 55 kDa subunit of human interleukin 2 receptor
  • anti-TFR-LysPE40 The activity of anti-TFR-LysPE40 was assayed on a variety of human cell lines and it inhibited protein synthesis in all the human cell lines studied (Table I). Anti-TFR-LysPE40 was most active on A431 cells with an ID 50 of 4.0 ng/ml. Specificity was demonstrated by showing that excess unconjugated antibody prevented the cytotoxic effect of anti-TFR-LysPE40 (Fig. 3A) . Ariti-TFR-LysPE40 was not cytotoxic to murine Swiss 3T3 cells even at 2 ⁇ g/ml (data not shown) .
  • anti-Tac-LysPE40 The cytotoxic activity of anti-Tac-LysPE40 was determined on HUT102 cells, a human T cell leukemia line containing IL2 receptors. As shown in Figure 5, anti-Tac- LysPE40 inhibited protein synthesis in HUT102 cells with an ID 50 of 2.5 ng/ml and excess anti-Tac blocked this effect demonstrating the specificity of the immunotoxin (Fig. 3B). Anti-Tac-LysPE40 did not inhibit protein synthesis on IL2 receptor negative cells, even at 2000 ng/ml, further showing the specificity of the IT. Blood levels of anti-TFR-LysPE40 in mice
  • mice were injected I.P. with a single dose of 100 ⁇ g of anti-TRF-LysPE40 and blood was drawn at different times after the injection to assay for immuno ⁇ toxin activity. As shown in Fig. 4, a peak blood level of 78 ⁇ g/ml was obtained 4 hrs after the injection and a level of 10 ⁇ g/ml was still present 24 hrs after the injection.
  • a similar experiment was performed in arthymic mice bearing A431 tumors. After injecting 50 ⁇ g of anti- TFR-LysPE40, a blood level of 27 ⁇ g/ml was detected 4 hrs after injection and 8 ⁇ g/ml after 24 hrs (Fig. 4). Effect of anti-TFR-LvsPE40 on A431 tumors in mice
  • Anti-TFR-LysPE40 was assayed for its ability to inhibit the growth of A431 cells as subcutaneous xeno- grafts in nude mice. To produce tumors, 3 x 10 6 A431 cells were injected subcutaneously on day 0. In the control group treated only with diluent, the tumors grew very rapidly and the animals were sacrificed on day 19 with very large tumors that were penetrating the skin (Fig. 5, 6). In a group that received anti-TFR-LysPE40 on days 2, 4, 6, 8, no tumors were evident on day 24 when the experiment was terminated (Fig. 5A and 6) . In another group of animals, treatment was delayed until the tumors were about 125 mm3 in volume and given on days 9, 11, 13 and 15.
  • LysPE40 can be efficiently coupled to antibodies yielding an immuno ⁇ toxin with high cytotoxic activity against cultured cell lines bearing the appropriate antigen, and no detectable cytotoxicity against cultured cells to which the antibody does not bind.
  • an immunotoxin composed of an antibody to the human transferrin coupled to LysPE40 (anti-TFR-LysPE40) could be administered safely in large amounts to mice and caused regression of a rapidly growing human epidermoid carcinoma implanted subcutaneously.
  • .tumor cells were allowed to grow to form a detectable solid tumor before treatment was initiated. Under these conditions, a treatment consisting of four injections given over eight days caused obvious tumor regression
  • a therapeutic composition in accordance with the present invention comprises an effective amount of the LysPE40-coupled to a target-specific ligand to kill target cells, and a pharmaceutically acceptable carrier, the target cells being those that are desired to be selective ⁇ ly killed and carry a binding site to which said ligand specifically binds.
  • ligands other than antibodies such as receptors, growth factors and molecules which selectively recognize target cells are coupled to the LysPE40 follow ⁇ ing the methodology similar to that described herein to obtain target-specific cytotoxic entities.
  • OVCAR4 30.0 ND MOLT4 32.0 ND a ID 50 is described as concentration of the immunotoxin needed for 50% inhibition of protein synthesis. N.D. - not done.
  • mice were injected subcutaneously with 3 x

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Abstract

Immunotoxine du conjugué LysPE40-anticorps, tuant sélectivement les cellules porteuses d'antigènes ou de récepteurs appropriés.
PCT/US1990/007464 1989-12-21 1990-12-21 Toxine amelioree d'elaboration d'immunotoxines WO1991009965A1 (fr)

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JP91502841A JPH04506976A (ja) 1989-12-21 1990-12-21 免疫毒素の構築のための改良された毒素

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US45416289A 1989-12-21 1989-12-21
US454,162 1989-12-21

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993010819A1 (fr) * 1991-11-26 1993-06-10 Alkermes, Inc. Procede de preparation de conjugues d'agents diagnostiques ou neuropharmaceutiques-anticorps specifiques de recepteur de transferine
US5527527A (en) * 1989-09-07 1996-06-18 Alkermes, Inc. Transferrin receptor specific antibody-neuropharmaceutical agent conjugates
US5571894A (en) * 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5672683A (en) * 1989-09-07 1997-09-30 Alkermes, Inc. Transferrin neuropharmaceutical agent fusion protein
US5939531A (en) * 1991-07-15 1999-08-17 Novartis Corp. Recombinant antibodies specific for a growth factor receptor
US5977307A (en) * 1989-09-07 1999-11-02 Alkermes, Inc. Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins
US6015555A (en) * 1995-05-19 2000-01-18 Alkermes, Inc. Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates
US6072035A (en) * 1992-09-25 2000-06-06 Novartis Corporation Reshaped monoclonal antibodies against an immunoglobulin isotype
US6329508B1 (en) 1989-09-07 2001-12-11 Alkermes, Inc. Transferrin receptor reactive chimeric antibodies
US8932586B2 (en) 2011-09-06 2015-01-13 Intrexon Corporation Modified forms of Pseudomonas exotoxin A
WO2016146833A1 (fr) 2015-03-19 2016-09-22 F. Hoffmann-La Roche Ag Biomarqueurs de résistance à la nad(+)-diphtamide adp-ribosyltransférase
EP3184547A1 (fr) 2015-10-29 2017-06-28 F. Hoffmann-La Roche AG Anticorps anti-tpbg et procédés d'utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4545985A (en) * 1984-01-26 1985-10-08 The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services Pseudomonas exotoxin conjugate immunotoxins
US4892827A (en) * 1986-09-24 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4545985A (en) * 1984-01-26 1985-10-08 The United States Of America As Represented By The Secretary, Dept. Of Health And Human Services Pseudomonas exotoxin conjugate immunotoxins
US4892827A (en) * 1986-09-24 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Cell, Volume 48, issued 16 January 1987, J. HWANG et al., "Functional Domains of Pseudomonas Exotoxin Identified by Deletion Analysis of the Gene Expressed in E. coli", pages 129-136, see entire document. *
Journal of Biological Chemistry Vol. 263, No. 19, issued 05 July 1988, T. KONDO et al., "Activity Immunotoxins Constructed with Modified Pseudomonas Exotoxin A Laking the Cell Recognition Domain", page 9470-9475, see entire document. *
Nucleic Acids Research, Volume 9 No. 15, issued July 1981, R. B. WALLACE et al., "Oligonucleotide directed mutagenesis of the human B-globin gene: a general method for producing specific point mutations in cloned DNA", pages 3647-3656, see the entire document. *
See also references of EP0506854A4 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5527527A (en) * 1989-09-07 1996-06-18 Alkermes, Inc. Transferrin receptor specific antibody-neuropharmaceutical agent conjugates
US5672683A (en) * 1989-09-07 1997-09-30 Alkermes, Inc. Transferrin neuropharmaceutical agent fusion protein
US5833988A (en) * 1989-09-07 1998-11-10 Alkermes, Inc. Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates
US5977307A (en) * 1989-09-07 1999-11-02 Alkermes, Inc. Transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins
US6329508B1 (en) 1989-09-07 2001-12-11 Alkermes, Inc. Transferrin receptor reactive chimeric antibodies
US5571894A (en) * 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5939531A (en) * 1991-07-15 1999-08-17 Novartis Corp. Recombinant antibodies specific for a growth factor receptor
WO1993010819A1 (fr) * 1991-11-26 1993-06-10 Alkermes, Inc. Procede de preparation de conjugues d'agents diagnostiques ou neuropharmaceutiques-anticorps specifiques de recepteur de transferine
US6072035A (en) * 1992-09-25 2000-06-06 Novartis Corporation Reshaped monoclonal antibodies against an immunoglobulin isotype
US6015555A (en) * 1995-05-19 2000-01-18 Alkermes, Inc. Transferrin receptor specific antibody-neuropharmaceutical or diagnostic agent conjugates
US8932586B2 (en) 2011-09-06 2015-01-13 Intrexon Corporation Modified forms of Pseudomonas exotoxin A
US9371517B2 (en) 2011-09-06 2016-06-21 Intrexon Corporation Modified forms of Pseudomonas exotoxin A
US9447387B2 (en) 2011-09-06 2016-09-20 Intrexon Corporation Modified forms of pseudomonas exotoxin A
US9677055B2 (en) 2011-09-06 2017-06-13 Interxon Corporation Modified forms of Pseudomonas exotoxin A
WO2016146833A1 (fr) 2015-03-19 2016-09-22 F. Hoffmann-La Roche Ag Biomarqueurs de résistance à la nad(+)-diphtamide adp-ribosyltransférase
EP3184547A1 (fr) 2015-10-29 2017-06-28 F. Hoffmann-La Roche AG Anticorps anti-tpbg et procédés d'utilisation

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CA2071946A1 (fr) 1991-06-22
AU636453B2 (en) 1993-04-29
JPH04506976A (ja) 1992-12-03
EP0506854A1 (fr) 1992-10-07
AU7172391A (en) 1991-07-24

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